مجله پژوهش های سلولی مولکولی (زیست شناسی ایران)
سال سی و دوم شماره 3 (پاییز 1398)

In the present study, an enzyme catalyzed in situ forming hydrogel based on chemically modified tragacanth was prepared and then evaluated for use in cartilage tissue engineering. For this purpose, firstly tyramine was conjugated on the galacturonic acid methyl ester units of gum tragacanth (GT) via ammonolysis of methyl ester groups in heterogeneous media. Then, the hydrogel was prepared by mixing of functionalized polymer, horseradish peroxidase (HRP) and hydrogen peroxide using a double syringe equipped with a mixing chamber. Then, cell viability of the encapsulated human mesenchymal stem cells (hMSCs) and in vitro chondrocyte differentiation of them, incubated in the presence of differentiation medium, were investigated. After mixing of the gel promoters, hydrogel formation was obtained due to enzyme catalyzed oxidative coupling reaction between phenolic groups of tyramine functional groups. The tunable gelation time of hydrogels was less than 2 minute. Viability of the encapsulated cells was more than 95% and 75% after 2 h and 21 days of incubation. The expression of chondrocytic genes and sulphated glycosaminoglycan indicated that the encapsulated hMSCs differentiated to chondrocyte cells during in vitro differentiation time.

Nerve growth factor (NGF) is a well-characterized member of the neurotrophin protein family, which has been shown to play a pivotal role in treating diseases such as multiple sclerosis and Alzheimer’s disease. Production of recombinant proteins in E. coli has particular disadvantages, which are primarily inclusion body formation and lack of disulfide bond formation in the cytoplasm. We therefore aimed to use the modified signal peptide of the Iranian native Bacillus licheniformis alpha-amylase to target the expressed recombinant β-NGF to the periplasm as an effective strategy to produce correctly-folded β-NGF. For this purpose, the human β-NGF gene with the signal sequence of the Iranian native Bacillus licheniformis alpha-amylase was cloned into a pET21a (+) vector and then the recombinant vector was transformed to BL21 (DE3) and BL21 (DE3) plysS strains. Protein expression was induced by 1mM IPTG in strains with the recombinant vector and the positive control strain containing the pET39b (+) vector with the DsbA signal peptide. Cytoplasmic and periplasmic expression of β-NGF was confirmed by SDS-PAGE and dot-blot assays. Finally, the expressed periplasmic proteins were purified using affinity chromatography and this purification was confirmed by using SDS-PAGE and Western blot assays. The results show the modified signal peptide of the Iranian native Bacillus licheniformis alpha-amylase is more effective for the secretory expression and directing β-NGF to the periplasmic space than the DsbA signal peptide. Also, these results indicate that the BL21 (DE3) plysS strain is an appropriate host for periplasmic expression of β-NGF.